Combining Single Crystal Electron Paramagnetic Resonance and X-Ray Crystallography to Study the Orientation and Dynamics of MTSSL Spin Labels in T4 Lysozyme

Abstract

The line shape of cw electron paramagnetic resonance (EPR) spectra of spin labeled proteins has been used extensively to analyze local dynamics, backbone fluctuations and structural rearrangements of these systems. The commonly used paramagnetic spin label MTSSL, a methanethiosulfonate derivative, attached to the protein via covalent disulfide coupling on a designated cysteine residue serves as a reporter of these properties. Much effort has been made to extract structural and dynamic information as encoded in the observed line shapes.In this study we use single crystals of MTSSL labeled T4 lysozyme as a prototype example of an α-helical protein to investigate the structure and dynamics of the paramagnetic side chain in detail by a combination of X-ray crystallography and EPR spectroscopy. X-ray diffraction is used to determine the orientation of the unit cell with respect to the EPR sample tube, which in turn allows to determine the orientation of the spin labels for every EPR spectrum of an angular dependent series given that the structure and orientation of the protein in the unit cell is known. To this end high-resolution crystal structures (0.9 - 1.3 A) of spin labeled T4 lysozyme were taken at 100 K and are compared to data deposited in the protein data bank.On this poster we will discuss the angular dependent line shape of the EPR spectra taken at room temperature with respect to the orientation of the spin label as deduced from X-ray crystallography at 100 K. Particular emphasize will be on the ability of currently available models to describe the observed dynamics of the spin label and the implications of this for the line shape analysis of disordered samples.